Improved capillary electrophoretic separation of glycosylated oligopeptides through addition of poly(vinyl alcohol), and analysis by electrospray mass spectrometry

A method for the analysis of O-glycosylation of peptides has been developed, combining capillary electrophoretic (CE) separation and electrospray ionization mass spectrometry. Synthetic peptides with apomucin 'tandem repeat' sequences which present potential O-glycosylation sites on threonine and serine residues were used as model system. In vitro O-glycosylated peptide samples were obtained by incubation of the peptides with human gastric microsomal homogenates containing N-acetylgalactosamine transferase activity in the presence of uridyl diphosphate N-acetylgalactosamine (UDP-GalNAc). CE was carried out in the presence of the linear polymer poly(vinyl alcohol) in the electrophoresis solvent, resulting in a greatly improved separation of the up to five different glycoforms of peptides with lengths of 8, 16 or 23 amino acids, and the unglycosylated peptides. After separation and peak collection, the number of modifications with N-acetyl galactosamine (GalNAc) could be determined by electrospray ionization mass spectrometry. The glycosylation pattern was shown to depend on the amino acid sequence of the peptides.